GenTarget`s EcoTMPlasmid DNA Miniprep Kit User Manual
Contents
1. 17 945 5033 or T 49 0 69 779099 F 44 0 1235 820 482 T 1 800 987 0985 F 49 0 69 13376880 F 1 617 945 8218 Switzerland Centro Nord Sud 2E CH 6934 Bioggio Lugano T 41 0 91 604 55 22 F 41 0 91 605 17 85 Related Products Product Product Description Category please Category name to see product s pages Fluorescent Premade Lentivirus for GFP CFP YFP RFP protein Human and Premade lentivirus expressing human and mouse ORFs with RFP Blasticidin fusion mouse ORFs dual markers CRE Premade lentivirus expressing nuclear permeant CRE recombinase with different recombinase flurescent and antibiotic markers LoxP Premade lentivirus expressing LoxP GFP Stop LoxP RFP cassette used to ColorSwitch monitor the CRE recombination event in vivo TetR inducible Premade lentivirus expressing TetR tetracycline regulator protein the repressor expression protein for the inducible expression system repressor Premde lentivirus for human and mouse iPS Myc NANOG OCT4 SOX2 FLF4 iPS factors factors with different fluorescent and antibiotic markers T antigen Expression of large and small T antigen with different selection markers Expression Cell Organelle Premade lentivirus for cell organelle imaging The fluorescent marker imaging GFP RFP CFP is localized in different cell organelles for live cell imaging LacZ expression Expression of full length B galactosidase lacZ with different2. Pre made Lentiviral Particles for target over expression manual for human mouse or rat genes ORFs Amount 200ul vial at gt 1 x 10 IFU ml or in PBS as special request Storage lt 70 C avoid repeat freeze thaw cycles Stable for 6 months at lt 70 C Product Description Lentiviral system is a gene delivery tool using lentivector for gene expression or knockdown Lentivector is HIV 1 Human Immunodeficiency Virus 1 derived plasmids It produces lentiviral particles lentivirus that are capable to transduce into broad range of mammalian cell types or organs including primary cells and non dividing cells both in vivo and in cell culture system and stably integrated into the transduced cell s genome independent of cell cycle for long term expression Thus lentivirus holds unique promise as gene transfer agents Pre made lentiviral particles for specific human or mouse gene are generated from optional inducible lentiviral system see vector scheme below Vector has adapted self inactivation feature in its 3 LTR which only generates replication incompetent particles The representation of optional inducible lentivector core scheme target RSV WPRE Each particle expresses a full sequence verified human mouse or rat target matching to CDS sequence of each target according to NCBI accession ID The human targets were natively expressed under tetracycline inducible suCMV promoter A blasticidin RFP fusion dual marker ex
3. al stock at room temperature Add appropriate amount of virus stock to obtain the desired MOI Return cells to 37 C CO2 incubator Try to avoid thaw freeze cycles for pre made lentivirus But if you cannot use all virus at once you still can re freeze the virus at 80 C for future use But virus titer will decrease by 10 for each re thaw Day 3 At 72hr after transduction check the transduction rate via fluorescent imaging with a suitable filter under fluorescent microscope or calculate the exact transduction rate via Flow Cytometry System FACS or any flow cytometry such as Guava machine Day 3 optional Transduced cells can be sorted via FACS and selected by its specific antibiotic A pilot experiment should be done to determine the antibiotic s kill curve for your specific cell line Refer to any literature on How to generate stable cell lines 2 Suspension cells transduction Protocols 1 Grow your cell in complete suspension culture medium shaking flask in CO incubator if necessary 2 Measure cell density When cell grow to 3 x 10 cell ml measure cell viability should be gt 90 then dilute cells into 1 x 10 cell ml in complete medium AMSBIO www amsbio com info amsbio com SUZ UK amp Rest of the World BE North America Germany Switzerland AES 184 Park Drive Milton Park _ 1035 Cambridge Street Bockenheimer Landstr 17 19 Centro Nord Sud 2E Abingdon OX14 4SE UK Cambridge MA 02141 60325 Fran
4. articles Please refer CDC and NIH s guidelines for more details regarding to safety issues References BioTechniques 38 891 894 June 2005 Biosci Biotechnol Biochem 68 3 565 5570 2004 Annu Rev Microbiol 1994 48 345 69 Microbiol Mol Biol Rev 2005 Jun 69 2 326 56 APPLIED AND ENVIRONMENTAL MICROBIOLOGY July 2005 p 3427 3432 Molecular amp Biochemical Parasitology 155 2007 167 171 Biosci Biotechnol Biochem 68 3 565 570 2004 NIH Guidelines for Biosafety Considerations for Research with Lentiviral Vectors link 10 CDC guidelines for Lab Biosafety levels link 000 SS OU ee NS Warranty THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol 279 No 5 Issue of January 30 pp 3212 3217 2004 This product is warranted to meet its quality as described when used accordance with its instructions Amsbio disclaims any implied warranty of this product for particular application In no event shall Amsbio be liable for any incidental or consequential damages in connection with the products Amsbio s sole remedy for breach of this warranty should be at Amsbio s option to replace the products Those products are provided for research use only AMSBIO www amsbio com info amsbio com SZ UK amp Rest of the World BE North America Germany 184 Park Drive Milton Park _ 1035 Cambridge Street Bockenheimer Landstr 17 19 Abingdon OX14 4SE UK Cambridge MA 02141 60325 Frankfurt Main T 44 0 1235 828 200 T 1 6
5. kfurt Main CH 6934 Bioggio Lugano T 44 0 1235 828 200 T 1 617 945 5033 or T 49 0 69 779099 T 41 0 91 604 55 22 F 44 0 1235 820 482 T 1 800 987 0985 F 49 0 69 13376880 F 41 0 91 605 17 85 F 1 617 945 8218 3 Transduction thaw lentiviral particles at room temperature Simply add premade lentiviral particle into the diluted cells at ratio of 50 to 100ul virus per 0 5 ml of cells Note depending on the cell type you may need to use more or less virus Grow cells in flask shaking in CO2 incubator 4 At 24 hours after transduction add equal amount of fresh medium containing related antibiotic Note each particles contain an antibiotic marker and the antibiotic amounts to use depends upon cell types Grow cell in CO incubator 5 At 72 hours after transduction check fluorescence under microscope or calculate the transduction efficiency using cell sorting machine like FACS or Guava machine 6 You can sort the fluorescent positive cells and maintain the antibiotic selection to generate stable cell lines Safety Precaution Amsbio lentiviral particles have adopted the most advanced lentiviral safety features using the third generation vectors with self inactivation SIN 3UTR and the premade lentivirus is replication incompetent However please use extra caution when using lentiviral particles Use the lentiviral particles in Bio safety II cabinet Ware gloves at all times when handling lentiviral p
6. orth America Germany Switzerland ESS 184 Park Drive Milton Park _ 1035 Cambridge Street Bockenheimer Landstr 17 19 Centro Nord Sud 2E Abingdon OX14 4SE UK Cambridge MA 02141 60325 Frankfurt Main CH 6934 Bioggio Lugano T 44 0 1235 828 200 T 1 617 945 5033 or T 49 0 69 779099 T 41 0 91 604 55 22 F 44 0 1235 820 482 T 1 800 987 0985 F 49 0 69 13376880 F 41 0 91 605 17 85 F 1 617 945 8218 Note 1 Depending on your specific needs you may design the transduction with different MOI for different levels of expression 2 Forsome cell lines you may add polybrene for transduction enhancement Transduction Protocols 1 Adhesive cells Transduction Protocols Note A quick transduction protocol add 50ul virus into one well of 24 well plate when cell density is at 50 75 72 hours after virus has been added no need to change medium visualize the positive cells under fluorescent microscope For stable cell line generation pass cells into antibiotic containing medium or sort the cells via fluorescent signal and then select the cells by antibiotic Day 0 Seed the desired cells in complete medium at appropriate density and incubate overnight Note at the time of transduction cells should be 50 75 confluent For example seed HeLa cells at 0 5 x 10 ml x 0 5ml in a well of a 24 well plate Day 1 Remove the culture medium Add fresh warm complete medium 0 5ml Thaw the Pre made lentivir
7. ovide the Negative control lentivirus for establishing controls for lentivirus treatment in a given cell line which also validates the specificity of any target expression effects The control virus to use is CMV Null RB which has the identical lentivector backbone as target expression virus but does not express any target except the same dual marker The ready to use particles are packaged in 293T cells and provided as 200ul aliquot Note the particles can be provided in PBS as special request Particles are safe and easy to use simply add into cultured cells or organs Each particle is validated in lot by lot basis and the target expression is guaranteed Transduction of pre made lentiviral particles Lentiviral Membrane particles fusion uncoating oa Translation Reverse evecievel expression CI integration Viral RNA a Key features 1 High target expression level driven by strong suCMV promoter 2 High virus titers that can be readily verified by the RFP fluorescent signal 3 Optional tetracycline inducible expression when desirable 4 Easy transduction monitoring via the RFP fluorescent signal under microscope 5 Dual markers transduced cells can be sorted via a RFP fluorescent signal or selected via blasticidin antibiotic 6 The lentivirus is ready and easy to use simply add into your cell culture see transduction carton image above AMSBIO www amsbio com info amsbio com SZ UK amp Rest of the World BE N
8. pressed under RSV promoter allows to sort or to select the transduced cells via RFP signal or via blasticidin antibiotic respectively RFP signal provides a convenient real time monitoring the particles performance However when inducible expression is desired they can optionally be used as tetracycline inducible expression in the presence of a repressor protein TetR tetracycline regulator protein For inducible expression the target expression is first repressed by TetR and induced after tetracycline addition The presence of TetR can be achieved by co infection of premade TetR lentiviral particles or co transfected with a TetR expression plasmid or simply by a tetR expressing stable cell line Please see our website for more AMSBIO www amsbio com info amsbio com SZ UK amp Rest of the World BE North America Germany Switzerland AS 184 Park Drive Milton Park _ 1035 Cambridge Street Bockenheimer Landstr 17 19 Centro Nord Sud 2E Abingdon OX14 4SE UK Cambridge MA 02141 60325 Frankfurt Main CH 6934 Bioggio Lugano T 44 0 1235 828 200 T 1 617 945 5033 or T 49 0 69 779099 T 41 0 91 604 55 22 F 44 0 1235 820 482 T 1 800 987 0985 F 49 0 69 13376880 F 41 0 91 605 17 85 F 1 617 945 8218 information about the inducible lentiviral system Amsbio provides the TetR lentiviral particles with different antibiotic selection marker for double selection of the inducible target expression cells We also pr
9. selection markers Fluorescent ORF fusion Pre made lentivirus for expression of GFP RFP CFP ORF fusion targets Pre made shRNA lentivirus Premade shRNA lentivirus for knockdown of a specific gene P53 LacZ Luciferase and more microRNA and anti microRNA Premade lentivirus expressing human or mouse precursor miRNA and anti miRNA lentivector and virus for human and mouse miRNA lentivirus Negative control Premade negative control lentivirus with different markers serves as a negative lentiviruses control of lentivirus treatment for validation of the specificity of any lentivirus target expression effects Other Enzyme expression Ready to use lentivirus expressing a specific enzymes with different selection markers AMSBIO www amsbio com info amsbio com AUZ UK amp Rest of the World AES 184 Park Drive Milton Park Abingdon OX14 4SE UK T 44 0 1235 828 200 F 44 0 1235 820 482 North America 1035 Cambridge Street Cambridge MA 02141 T 1 617 945 5033 or T 1 800 987 0985 F 1 617 945 8218 German Bockenheimer Landstr 17 19 60325 Frankfurt Main T 49 0 69 779099 F 49 0 69 13376880 Switzerland Centro Nord Sud 2E CH 6934 Bioggio Lugano T 41 0 91 604 55 22 F 41 0 91 605 17 85
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